Hemophilia is a group of hereditary genetic disorders that impair the body's ability to control blood clotting or coagulation. In its most common form, Hemophilia A, clotting FVIII is deficient, Hemophilia A occurs in about 1 in 5,000-10,000 male births. The FVIII protein is an essential cofactor in blood coagulation with multifunctional properties. The deficiency of FVIII can be treated with plasma-derived concentrates of FVIII or with recombinantly produced FVIII. The treatment with FVIII concentrates has led to a normalized life of the hemophilia patients. Historically, Hemophilia A has been treated with FVIII originating from human blood plasma. In blood plasma, under normal conditions, the FVIII molecule is always associated with its cofactor; von Willebrandt factor (vWf), which stabilizes the FVIII molecule from different forms of degeneration.
Plasma derived FVIII products occur on the market with different purities and with more or less amounts of vWf present. Commonly, products with low amount of vWf contain added human albumin and or other stabilizers including increased salt concentration to stabilize the FVIII molecule). The methods used to purify FVIII were normally a combination of different precipitation methods such as cryo precipitation, aluminum hydroxide precipitation etc. and chromatography steps mainly ion exchange, affinity and gel filtration steps.
In order to improve FVIII products affinity chromatography was employed, which effectively removed contaminants to a high degree of FVIII purity including the possibility to reduce also vWf (Farrugia et al., Biotechnology and plasma fractionation industry; The impact of advances in the production of coagulation FVIII. Biotechnology, Vol. 3, No. 1, February 1993). The disadvantage with immuno affinity chromatography was that it is relatively expensive and that the monoclonal antibodies used as affinity ligands, where of animal origin.
In the mid 80's there where some virus transmissions associated with plasma derived FVIII products. Even if this problem was solved through implementation of specific virus reduction steps, this was the starting point of the development of recombinant FVIII products (rFVIII). In the 90's, the first rFVIII product was marketed and up to date there are three different rFVIII products (two full length molecules and one B-domain deleted molecule in which an inactive part of the FVIII molecule has been removed to increase the productivity of the host cell (Eriksson et al., The manufacturing process for B-domain deleted recombinant FVIII. Seminars in Hematology, Vol 38, No 2, Suppl. 4 (April), 2001: pp 24-31)) with a high degree of purity (all without vWf).
The purification methods used to purify the rFVIII, all were a combination of different chromatography techniques (ref. Bhattacharyya et al., Review article; Recombinant FVIII for Haemophilia “An overview of production technologies”. CRIPS Vol. 4, No. 3, July-September 2003). One was the known immuno affinity technique (even if there are products solving this, for example with peptide affinity (Kelly et al., Development and validation of an affinity chromatography step using a peptide ligand for cGMP production of FVIII.) or a yeast derived antibody fragment (VIIISelect FVIII affinity resin—GE Healthcare, Cat. No. 17-5450 presently are entering the market) as used for the plasma FVIII.
As vWf is absent in all rFVIII products, certain measures have to be taken to stabilize the FVIII molecule against loss of activity (aggregation, proteases, surface adsorption etc.). In one of the products, a chelating agent (EDTA etc.) is added to protect FVIII against degeneration of metallo proteases (U.S. Pat. No. 5,831,026). To add albumin, aprotinin, insulin or even to co-express rFVIII with vWf (and remove it down stream in the purification cycle) are strategies which have been performed to increase the stability of the rFVIII molecule (ref. Bhattacharyya et al., Review article; Recombinant FVIII for Haemophilia “An overview of production technologies”. CRIPS Vol. 4, No. 3, July-September 2003).
Another strategy (to maintain a process free of mammalian additives and chelating agents) is described in EP-A-1 707 634, where a combination of increased amounts of salts, contribute to the stability and high recovery of the rFVIII product (Wang et.al, Coagulation FVIII, structure and stability. International Journal of Pharmaceuticals, 259 (2003), 1-15.). However, this technique has a certain disadvantage. For example, the relatively high salt content makes it not suitable to process directly to an ion exchanger without dilution (and possible destabilization Parti et al., In vitro stability of recombinant FVIII. Haemophilia (2000), 6, 513-522. Biotechnology and Bioengineering, Vol. 87, No. 3, Aug. 5, 2004.).
WO-A-2009/007451 discloses a purification method of FVIII using a mixed-mode or multimodal resin. The purification method is based on contacting FVIII protein with a multimodal or mixed-mode resin containing ligands which comprise a hydrophobic part and a negatively charged part and eluting said FVIII protein with an elution buffer containing at least 1.5 M salt and at least 40% (w/v) of ethylene glycol, propylene glycol or a mixture thereof, and calcium ions.
EP-A-1707634 discloses a method for isolation of recombinantly produced proteins i.a. by various methods such as immuno-affinity chromatography, affinity chromatography, protein precipitation, buffer exchanges, ionic exchange chromatography, hydrophobic interaction chromatography, mixed-mode hydrophobic/ion exchange chromatography media, chelating chromatography, carbohydrate affinity like lectin or heparin affinity chromatography, size-exclusion chromatography, electrophoresis, dialysis, different precipitation agents such as polyethylene glycol, ammonium sulphate, ethanol, hydroxy apatite adsorption, filter membrane adsorption, ligands coupled to magnetic particles etc. However, it is identifying particular chromatographic purification steps.
WO-A-2005-082483 discloses a process for the purification of antibodies from one or more impurities in a liquid, which process comprises contacting said liquid with a first chromatography resin comprised of a support to which multimodal ligands have been immobilised to adsorb the antibodies to the resin, wherein each multi-modal ligand comprises at least one cation-exchanging group and at least one aromatic or heteroaromatic ring system. An eluant is added to release the antibodies from the resin and the eluate is contacted with a second chromatography resin.
WO-A-2005/121163 discloses a process for the isolation of one or more proteins from a protein solution. The process comprises the steps of providing a protein solution comprising one or more specific proteins and having a preset pH and a preset ionic strength or conductivity, applying the protein solution to a packed bed or expanded bed column comprising an adsorbent, and obtaining one or more proteins fromt he column, wherein the protein solution has been supplemented with an alcohol.